Glucocorticoid hormones

As early as in the 1930s, the hormone cortisone was isolated from the adrenal glands and its efficacy for treatment of rheumatoid arthritis was empirically demonstrated in patients suffering from this debilitating disease. Adrenal corticosteroids are any of the steroid hormones produced by the adrenal cortex except for the sex hormones. These include the mineralocorticoids (aldosterone) and glucocorticoids (cortisol), the secretion of which is regulated by the adrenocorticotrophic hormone (ACTH) produced in the anterior pituitary. Due to their immunosuppressive effects, corticosteroids or glucocorticoid hormones (GCs) are used to reduce organ rejection after transplantation and to treat auto-immune diseases, including multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease. Other inflammatory diseases for which GCs make part of the standard treatment are systemic lupus erythematosus (SLE), sarcoidosis, asthma (Barnes and Adcock, 1997) and atopy (Beyaert, 1999). They are also used to treat brain edema, shock conditions, certain types of blood cancer (B- and T-cell lymphoma) (Jehn and Osborne, 1997), as well as conditions involving adrenal cortex insufficiency. GCs inhibit leukocyte migration to sites of inflammation and thus reduce the general symptoms of inflammation (Cato and Wade, 1996). The synthesis and secretion of cortisol, the naturally occurring glucocorticoid in humans, is subject to a negative feedback loop and under tight control by a careful balance between adrenocorticotropin hormone, secreted from the pituitary gland in the brain and corticotropin hormone, secreted from the hypothalamus in a pulsatile and circadian fashion (Balsalobre et al., 2000). GCs exert their functions through binding to the glucocorticoid receptor (GR), a transcription factor that can regulate genes in a positive or negative way. Transcription factors are proteins that are able to bind the promoter regions of their target genes and can modulate the rate of gene transcription. In a resting cell they can either already be in the nucleus bound to DNA and waiting to be activated (e.g. by a phosphorylating signal), or they can reside in the cytoplasm kept in an inactive state before activation allows them to travel inside the nucleus. Just as NF-κB and its set of specific inhibitor molecules ΙκΒ, inactive GR is sequestered in the cytoplasm, although not via a specific inhibitor but through its interaction with chaperoning molecules including Hsp90 and Hsp70. Because of their lipophilic nature, ligands for GR (cortisol, corticosterone or the synthetic dexamethasone DEX) can travel through the cell membrane, circumventing the need for membrane receptors to transmit their signal. Ligand binding induces a conformational change in GR, causing the release of the interacting molecules and exposing its nuclear localisation signal (a stretch of basic residus recognized by importin nuclear transport proteins).

Once in the nucleus GR can bind as a homodimer onto the glucocorticoid response element (GRE), an imperfect palindromic recognition sequence GGTACAnnnTGTYCT (Y=T or C), and positively regulate steroid-responsive genes regulating metabolic homeostasis. The GR protein consists of three main protein modules, an N-terminal heavily phosphorylated transactivating domain, a C-terminal dimerisation and ligand binding domain (LBD) and a central DNA-binding domain (DBD) of which eight out of nine cysteins form two tetrahedric structures in which a Zn ion is held. This special structure mediates contact with the DNA; GR is therefore also called a Zinc finger protein. Although these 3 modules can work quasi independently of each other, there is a great overlap in certain functions. For example, transactivation functions are also found in the DBD and LBD and nuclear translocation is not only found back in the LBD but also in the DBD. GR can also influence gene expression indirectly. A recently recognized important function of activated GR is the inhibition of transcription of several cytokines and chemokines that are relevant in inflammatory diseases. In the last decade it has become evident that one of the main targets for GCmediated cytokine gene suppression is NF-κB. Recent data, derived from elegant studies with dimerisationdefective (and hence transactivation-defective since only a dimeric GR recognizes the GRE consensus sequence) knocked-in GR mutant receptors in mice, demonstrated that the anti-inflammatory action of GCs solely arises from the ‘negative’ cross-talk of GR with NF-κB or AP-1 (Reichardt et al., 1998; Reichardt et al., 2001). The interaction between different transcription factors resulting in either cooperative enhancement or inhibition of gene expression is referred to as ‘cross-talk’. This is an important concept, as cross-talk provides an additional platform of regulation to increase the number of possible gene-specific responses in a cell-specific manner. The long-term use of GCs in patients with chronic inflammatory disorders is, sadly enough, overshadowed by severe metabolic side effects, ascribed to the transactivating function of GR. Endogenous GCs protect the body from stress by regulating blood pressure levels, blood glucose levels, liver glycogen deposition and lipid metabolism. Consequently, a long-term treatment with glucocorticoids can result in diabetes, redistribution of fat and hypertension, but also HPA axis (hypothalamo pituitary adrenal axis) insufficiency, osteoporosis, skin and muscle atrophy, increased susceptibility to infections, cataract, peptic ulcers and a general retention of water due to a disturbed water household balance (because high levels of GCs can also activate the mineralocorticoid receptor) (Boumpas et al., 1993; Karin, 1998). Besides its metabolic actions, GCs also affect brain functions such as behavior and memory (De Kloet et al., 1998); long-term steroid use may therefore also lead to neuropsychiatric conditions. An additional problem is the fact that patients treated with GCs for long periods may develop a resistance towards a steroid-based therapy (Barnes, Greening and Crompton, 1995). For all those reasons, the quest for ‘better’ anti-inflammatory drugs, separating the beneficial from the detrimental effects (so-called dissociated GCs) is a vigorous one. Progress has been made with the recent development and characterization of ‘dissociating’ glucocorticoids, which can separate to a certain extent transrepression from transactivation functions of GR (Resche-Rigon and Gronemeyer, 1998; Vanden Berghe et al., 1998). In order to improve further therapies for inflammatory disorders, the understanding of the molecular action mechanism of GCs is an absolute prerequisite. By no means a consensus has been reached with regard to the mechanism deployed by GCs in mediating pro-inflammatory gene repression. Different models aiming to explain this phenomenon have been put forward and will be discussed below.

Cytoplasmic models

1. Upregulation of IκB-α by glucocorticoids

One way GCs could repress NF-κB-driven gene expression is by sequestration of NF-κB in the cytoplasm. In the resting state, NF-κB activation is tightly controlled by its cytoplasmic inhibitor, IκB-α, which associates with NF-κB and prevents its migration to the nucleus. Two independent research groups proposed that GCs induced IκB-α. This newly made IκB-α would then travel to the nucleus to capture NF- κB from the DNA, ultimately resulting in retention of NF-κB in the cytoplasm and thus inhibition of cytokine gene expression (Auphan et al., 1995; Scheinman et al., 1995a). This mechanism was demonstrated for monocytes and T cells, but other groups including ours did not find evidence for this mechanism in other cell lines including lung epithelial, fibroblasts and endothelial cells (Adcock et al., 1999; Brostjan et al., 1996; De Bosscher et al., 1997; Hofmann et al., 1998). Actually, in many cases GCmediated repression occurred without a concomitant decrease in the DNA-binding capacity of NF-κB, as determined by gel retardation assays (Brostjan et al., 1996; De Bosscher et al., 1997; Newton et al., 1998). On top of that, in some cases IκB-α was found to be upregulated upon GC treatment but quite unexpectedly also without an apparent loss or decrease of NF-κB binding (Lezoualc'h et al., 1998; Wissink et al., 1998). It was recently shown for the ICAM-1 gene by in vivo footprinting analysis that GC repression happens without a change in conformation of the protein complex that binds to the NF-κB binding site (Liden et al., 2000). It thus seems that the upregulation of IκB-α by glucocorticoids is a cell-specific phenomenon first observed for monocytes and T-lymphocytes (Auphan et al., 1995; Scheinman et al., 1995a). However, this observation is not unique or universal to immune cells. For instance, GCs do not upregulate IκB-α in CD4+ cells in vivo (Reichardt et al., 2001), whereas in cell types, such as PC12, hepatocytes and breast carcinoma cells, GCs do cause an increase in IκB-α protein levels (De Vera et al., 1997; Lezoualc'h et al., 1998; Ray and Searle, 1997).

An interesting finding proving the point of cell specificity is that in the neuronal cortex of DEX-treated rats no IκB-α was upregulated, whilst in the peripheral cells of the same animal IκB-α levels were found to be enhanced (Unlap and Jope, 1997). Another example reflecting differences between different cell types are exemplified by the following observations. Elevated levels of IκB-α were found in vascular endothelial tissue but not in mononuclear cell infiltrates from GC-treated patients with Crohn’s disease. Note that this result also seems to clash with the in vitro data, in which mainly T-cells seemed to respond to GCs with a higher IκB-α protein level (Thiele et al., 1999). As an IκB-α promoter construct was unresponsive to upregulation by DEX in L929sA cells, the actual mechanism by which GCs enhance IκB-α levels in other cell types is even more puzzling (Vanden Berghe et al., 1999b). Establishing the upregulation of IκB-α by GCs is one thing but the critical question is whether this effect can also be linked to the GC-repression of NF-κB driven genes.The answer is negative. Following observations have clearly pointed out that IκB-α upregulation by GCs can be uncoupled from their anti-inflammatory cytokine gene-repressive effects. Mutational analysis of GR has shown that its DNA-binding capacity is dispensable for mediating transrepression on NF-κB-driven genes (Caldenhoven et al., 1995). This is confirmed in vivo by experiments using mice with a knocked-in dimerisation-defective GR dim/dim mutant (A458T), which has lost DNA-binding and gene-activating properties (Reichardt et al., 1998). Vice versa, a GR mutant (S425G) defective in NF-κB targeted gene repression was shown to still be capable of mediating enhanced IκB-α synthesis (Tao, Williams-Skipp and Scheinman, 2001). The repressive effects of GCs further remained apparent in the presence of the protein synthesis inhibitor cycloheximide, presenting evidence that novel protein synthesis is not required to inhibit NF-κB driven gene expression (De Bosscher et al., 1997; Wissink et al., 1998). Finally, the use of ‘dissociated’ compounds which lack GR transactivating capacities demonstrated that GR-mediated transcription is not required for the inhibition of p65 transactivation and, reciprocally, GC analogues which lack anti-inflammatory properties in vivo could still upregulate IκB-α (Heck et al., 1997; Vanden Berghe et al., 1999b).

The question remains why in some cell types IκB-α is upregulated by GCs and in other not. One possible explanation could be a functional difference with regard to apoptosis. NF-κB has been reported to have an anti-apoptotic function, as evidenced by the high level of liver apoptosis and subsequent death observed in p65-knockout embryos (Beg et al., 1995). Furthermore, NF-κB transcriptional activity has been implicated in cell cycle progression (Hinz et al., 1999; Kaltschmidt et al., 1999). In contrast, normal T-lymphoid and monocytic cells are sensitive to GC-induced apoptosis, a characteristic of GR used to its advantage in lymphoid cancers. Now, T-cells of GR dim/dim mice were no longer subject to GC-mediated apoptosis, arguing for the need of gene inductive effects by GR to mediate this event. IκB-α induction was found in a GC-induced apoptosis-sensitive cell line, but not in resistant human leukemic T cells (Ramdas and Harmon, 1998). Taken together, in lymphoid cells (which contain a high constitutive level of protective NF-κB) or other cells that have suffered too much damage, the IκB-α upregulation by GCs may ensure a functional apoptotic program to limit systemic immune responses that can otherwise lead to a lethal shock of the whole organism.

2. Interference by other signal transduction pathways

A completely different mechanism by which GCs may exert part of their anti-inflammatory effects is the inhibition of signalling pathways that regulate inflammatory processes. One such example is the extracellular regulated kinase ERK-1,2, controlling the release of allergic mediators and induction of proinflammatory cytokine gene expression in mast cells. Recently, the mechanism by which glucocorticoids inhibit ERK kinase activity was unraveled. This involves the increased expression and, crucially, at the same time a diminished proteosomal degradation of MAP kinase phosphatase-1 (Kassel et al., 2001). In other cell lines however, such as L929sA mouse fibroblasts, GCs did not inhibit tumor necrosis factor (TNF)-activated ERK activity (De Bosscher, Vanden Berghe and Haegeman, 2001). The lack of blockage of MKP-1 degradation by GCs in these cells, as observed for NIH3T3 mouse fibroblasts (Kassel et al., 2001), is probably responsible for the differential outcome. The anti-inflammatory action of glucocorticoids may therefore not only be due to negative regulation by GR, but can also involve positive regulation by this receptor. Other examples of anti-inflammatory proteins upregulated by GCs include secretory leukocyte protease inhibitor, which protects healthy lung tissue from leukocytes activated during airway inflammation (Abbinante-Nissen, Simpson and Leikauf, 1995), β-4-sulfoxide, which has a potent anti-inflammatory action on monocytes and macrophages (Young et al., 1999), IL1-receptor antagonist and lipocortin I and II, which are phospholipase inhibitor proteins (Barnes, 1998). Although, lipocortin upregulation by GCs may rather be a tissue or cell-specific effect since GCs did not affect its synthesis in L929sA fibroblasts and could therefore not be held responsible for the protective effects of DEX to TNF-mediated cytotoxicity (Beyaert et al., 1990). Along the same line, GC-mediated inhibition of c-Jun N-terminal kinase (JNK) activity leads to inhibition of c-Jun phosphorylation. This helps explaining the repressive action of GR towards the activity of AP-1, another transcription factor involved in pro-inflammatory gene expression (Caelles, Gonzalez Sanch and Muñoz, 1997; De Bosscher et al., 2001; Swantek, Cobb and Geppert, 1997). Similar results have been found for the inhibition of p38 MAPK activity by GCs, although this again seems not to be a universal mechanism (De Bosscher et al., 2001; Lasa et al., 2001). The inhibition of p38 activation requires de novo protein synthesis (Lasa et al., 2001); in contrast, it was shown that GC-mediated repression of TNF-induced IL-6 mRNA occurred in the presence of cycloheximide, a protein synthesis inhibitor (De Bosscher et al., 1997). These differences may reflect subtle regulations by GR to enhance its repressive capacity over a longer period and again emphasize the diversity of regulatory possibilities GR has at hand to modulate cellular signalling events. Another type of signalling cascade reported to have an effect on NF-κB and/or GR-driven transactivation is the protein kinase A (PKA) pathway. NF-κB-driven transcription is regulated through phosphorylation of RelA by PKA. Phosphorylation of Ser276 in p65 was shown to be essential for complex formation with the coactivator molecule CBP (cAMP response element binding (CREB)-binding protein) and subsequent stimulation of transactivation (Zhong, Voll and Ghosh, 1998). The catalytic subunit of PKA (PKAc) is also able to potentiate GR-dependent transcription. Phosphorylation of a serine residu (Ser 276) in the RHD by PKA was further demonstrated to be essential for p65-mediated repression of GR transactivation. Mutation of p65 at this conserved PKA phosphorylation site abolished the potential of p65 to repress GR (Doucas et al., 2000). Strikingly, an exclusively cytoplasmic variant of p65 (obtained by deleting the NLS of p65) was still capable of mediating transrepression of a GR-activated mouse mammary tumor virus promoter-driven reporter gene. From this result it was concluded that targeting GC-driven gene expression by p65 occurs in the cytoplasm, involving PKAc-dependent signalling as the molecular interface of this inhibition (Doucas et al., 2000) For the reciprocal mechanism controversy arises. A GR deletion variant (amino acids 589-697 deleted) with a predominant cytoplasmic localization would also still inhibit NF-κB driven gene expression, arguing that a competition for PKAc would mediate mutual cross-coupling in the cytoplasm (Doucas et al., 2000). Mapping GR functions has however demonstrated that there is more than one NLS in GR (Beato, 1989; McEwan, Wright and Gustafsson, 1997). It can therefore not be ruled out that a minor proportion of this variant can still travel in and out of the nucleus, using the other NLS, and mediate gene repression in the nucleus. Furthermore, Ser276 was found not to be a key player for repression of NF-κB activity by GR. In this case, mutation of Ser276 to a Cysteine residu in a Gal4-p65 fusion protein did not affect the ability of GR to block NF-κB-driven transcription (De Bosscher et al., 2000b) (the use of Gal4 fusion proteins is discussed in more detail in the section below). It becomes more and more clear that the reciprocal mechanisms of transrepression, i.e. the mutual antagonistic effects of NF-κB and glucocorticoids, do not necessarily use the same molecular mechanisms.

Nuclear models:

1. Direct protein-protein interaction:

Direct binding of GR to DNA via a so-called ‘nGRE’ and as such negatively regulating gene expression is quite a rare event. One example of this is the osteocalcin gene. The osteocalcin promoter region to which GR binds is partially overlapping with the TATA box, occluding the build-up of a functional transcriptional complex (Meyer, Carlstedt Duke and Starr, 1997). In spite of the ability of glucocorticoids to induce gene transcription, the major anti-inflammatory effects of GCs occur through repression of inflammatory and immune genes that are driven by NF-κB or another mitogenic transcription factor, AP-1 (Adcock and Caramori, 2001). Intriguingly, the transrepressive relationship between GR and NF-κB appears to be mutual so it seemed most logic at the time that both proteins would actively hinder each other’s transactivating functions by no other means than a direct physical contact. The model of a direct interaction between GR and NF-κB was supported by several research groups for the last 5 years, however, only recently an actual physical interaction with endogenous proteins was demonstrated in A549 lung carcinoma cells (Adcock, Newton and Barnes, 1997). Some research groups have focused on mapping the involved functional domains in transrepression; albeit with sometimes conflicting results. For GR, domain swapping of its modular parts (N-terminal domain, DNA-binding domain (DBD) and ligand binding domain (LBD)) with other members of the nuclear receptor superfamily such as estrogen receptor (ER) and thyroid receptor (TR) demonstrated that the DBD was indispensable, not only for transactivation (as expected) but also for transrepression (Liden et al., 1997; Moras and Gronemeyer, 1998; Ray et al., 1997). To discern whether the DNA binding function of GR per se was crucial for NF-κB transrepression point mutations in the P-box (=DNA interacting N-terminal Zn finger) were performed. The outcome hereof was that GR binding onto a classical GRE could be disrupted, but the transrepressive properties on NF-κB or AP-1 remained untouched (Caldenhoven et al., 1995; Heck et al., 1994; Helmberg et al., 1995).

Another study, however, pointed to the other Zn finger, C-terminally placed, as a crucial determinant for NF-κB transrepression (Liden et al., 1997). The reason for this discrepancy remains undetermined but may reside in the nature of the investigated NF-κB response elements (e.g. binding different NF-κB family members) in different cell lines. It is equally possible that a different cofactor context (see further) or GR function-modulating chaperones in different cell lines may be contributing to these discrepancies. Recently, it became apparent that the promoter context may codetermine whether or not a specific nuclear receptor can interfere with NF-κB activity (Amrani, Lazaar and Panettieri, 1999; De Bosscher, Vanden Berghe and Haegeman, 2000a). Replacing the LBD with a non-related inert β-galactosidase moiety (to diminish unwanted effects of an incorrect folding of deletion variants), did not affect transrepression, arguing for a purely steric role of the ligand binding domain (Oro, Hollenberg and Evans, 1988). Conversely, also the domains of p65 involved in repression of GR activity have been mapped. Elaborate mutational analysis demonstrated that both the N-terminal Rel homology domain (RHD), containing DNA binding and dimerisation functions, and the C-terminal domain of p65, harbouring transactivation functions, are required for the repression of GR activity. However, in vitro a physical interaction was only observed between the RHD of p65 and GR (Scheinman et al., 1995b; Wissink et al. 1997).

A greater part of the gathered data points out that the protein-protein interaction between GR and NF-κB is most likely to occur in the nucleus. A first piece of evidence has been referred to above, namely the unchanged footprinting pattern of NF-κB bound to its response element in the GC-repressible ICAM promoter (Liden et al., 2000). Secondly, when p65 is fused to a DNA-binding yeast protein Gal4 this fusion protein is completely nuclear and now able to transactivate a GAL4 binding site containing reporter gene. Importantly, GCs can inhibit the transactivation of Gal4-p65 to the same extent as of wild-type p65, arguing that GC repression is a nuclear phenomenon (De Bosscher et al., 1997). The transactivating Cterminus of p65 has been shown to contact the general transcription factors TFIIB and TBP (TATA-binding protein) in vitro (Schmitz et al., 1995); this binding could help stabilizing TFIID interactions to build up a functional promoter initiation complex (PIC) and to start gene transcription. The importance of the promoter context close to the TATA box sequences in mediating transrepression was exemplified by the finding of a NF-κB-driven gene that was no longer responsive to GC-mediated repression (De Bosscher et al., 2000b). Finding the sequences that determine this unexpected promoter specificity is an important issue. Another study exploring events around the start site of transcription came up with exciting new evidence that GR mediates repression by interfering with the phosphorylation of a Serine residu in the Cterminal domain of RNA polymerase II, again, without inhibiting assembly of the PIC (Nissen and Yamamoto, 2000). Cofactors are nuclear proteins that are able to form a bridge between transcription factors and the basal transcription machinery, without contacting the DNA themselves (Horwitz et al., 1996). They are often associated with an enzymatic activity, either a histone acetylase or a histone deacetylase activity, depending on their function as a coactivator or a corepressor, respectively. Histone acetylation is believed to be important for relaxing chromatin and favouring gene transcription, the opposite holding true for histone deacetylation (Wolffe and Pruss, 1996; Wolffe, 1997). The abovementioned result postulates the existence of a novel type of corepressor, associated with the LBD of GR, possibly a serine-2- phosphatase or a serine-2 kinase inhibitor (Nissen and Yamamoto, 2000).

A logic question now is whether a cytoplasmic model is in any way reconcilable with a nuclear model. Different findings, dependant on which cell lines are used, have sometimes led to seemingly conflicting results. However, it is quite acceptable that a different constitution in cofactor complexes or a different subset of specific responsive target genes may integrate all the signals coming from the cytoplasm with subtle differences, thus generating a slightly different mechanistic response by GCs. Also, a dosage effect or the duration of the pro-inflammatory insult to the cells may be of importance to the way GCs go about to inhibit the signalling mediated by NF-κB. Besides the transcriptional effects discussed here, important GC effects have also been detected at the posttranscriptional level, such as mRNA destabilization of proinflammatory genes (viz. INOS, TNFα, GM-CSF, COX-2) (Chaudhary and Avioli, 1996; Delany, Gabbitas and Canalis, 1995; Lasa et al., 2001; Tobler et al., 1992). Functional GR must therefore be considered more as context-dependent, multi-targeting effectors, rather than as mediators of repression via one exclusive pathway.

2. Cofactor models:

As mentioned in the paragraph above, cofactor molecules provide an extra layer of transcriptional regulation in the nucleus. Coactivators are generally associated with a HAT (histone acetylase) activity, corepressors are associated with a HDAC (histone deacetylase) activity (Wolffe, wong and Pruss, 1997). The LBD of GR has been shown to interact, in a ligand-dependent way, with coactivators such as CBP/p300, GRIP1 and SRC-1 (Chakravarti et al., 1996; Eggert et al., 1995; Onate et al., 1995). The same coactivators have also been implicated in bridging other transcription factors, such as NF-κB and AP-1 to the basal transcription machinery (Gerritsen et al., 1997; Kamei et al., 1996; Perkins, 1997; Sheppard et al., 1998; Vanden Berghe et al., 1999a). Gene repression could therefore result from a competition between transcription factors for limiting amounts of coactivator molecules. A competition between p65 and GR for limiting amounts of CBP or SRC-1 was proposed to account for transrepression of NF-κB-dependent genes (McKay and Cidlowski, 1998; Sheppard et al., 1998). It is, however, difficult to understand how this mechanism would generate a specific transrepression of GC-repressible NF-κB- or AP-1-driven target genes only, since a great number of other transcription factors utilize CBP/p300 or SRC-1 as well for enhancing their transactivation properties. Amongst these are e.g. CREB, ATF-2, MyoD, p53, Tax an STAT-2 (reviewed in (Horwitz et al., 1996; Xu, Glass and Rosenfeld, 1999)), although preferences and difference have been noted. So, it has been reported that NF-κB-mediated transactivation requires the presence of CBP, SRC-1 and p/CAF, using mainly the HAT activity of p/CAF, while CREB for instance uses CBP, p/CIP and p/CAF, but not SRC-1 (Korzus et al., 1998; Sheppard et al., 1999). It may be that a constitution of different coactivator complexes provides a platform for the specific recognition and repression by GCs of certain transcription factor families only. Nevertheless, other experimentations argue against a cofactor squelching model to explain mutual transrepression between NF-κB and GR. Increasing the coactivator levels in the cell by transient overexpression generates a general dose-responsive increase in gene expression levels of NF-κB-driven gene expression. However, upon activating GR the relative repression levels remain unaffected. Furthermore, in case of a competitive mechanism one would expect that the interaction between p65 and CBP would be diminished or completely lost upon interaction with activated GR. This proves not to be the case (De Bosscher et al., 2000a). Additional evidence diminishing the role of CBP in transrepression is the fact that the GAL4 transactivating Gal4-p65 Ser276Cys mutant, leading to a defective CBP recruitment and subsequent loss of TNF inducibility, still demonstrated a functional repression by GCs (De Bosscher et al., 2000b). For the reciprocal mechanism, p65 mutation in the DNA-binding domain but with the predicted coactivator recruitment domains intact could no longer repress GC-mediated transactivation (Wissink et al., 1997). Finally, from the fact that dissociating ligands or GR point mutants can distinguish between transactivation and transrepression it can be deducted that GR is not always associated with the same cofactor surrounding. In fact, a different steric conformation of GR could lie at the basis for this phenomenon, achieving transrepression, when GR adopts a monomeric conformation as opposed to allowing transactivation of target genes, when GR is in a DNA-bound dimeric conformation (Lefstin and Yamamoto, 1998; Reichardt et al., 2001). These findings demonstrate an incompatibility with a general cofactor competiton model.

It must be noted that all the abovementioned experimental approaches depending on overexpression and microinjection overload the cell with transcriptional components, disregarding the influence of nuclear architecture and specific nuclear matrix targeting. This latter phenomenon is a concept that has recently gained importance. Territorial subdivision of transcription factor complexes in the nucleus (Doucas et al., 1999; Stenoien et al., 2000; Stewart and Crabtree, 2000) may explain how cofactors only target a certain gene in a designated compartment in the nucleus whilst leaving the same factors associated with different genes in other compartments intact (Francastel et al., 2000; Hager et al., 2000; Lemon and Tjian, 2000). A specific nuclear matrix targeting signal has been described to include parts of the DBD and transactivation domains of GR (DeFranco and Guerrero, 2000). Some members of the nuclear hormone receptor superfamily, such as Retinoic/Retinoid X Acid Receptors RAR/RXR and Thyroid Receptors (TR) are already bound to DNA in absence of ligand. In this particular situation corepressor complexes actively silence gene expression (Burke and Baniahmad, 2000). A HDACcontaining corepressor complex consisting of the components NcoR (nuclear corepressor)/SMRT (silencing mediator of retinoid and thyroid receptors), mSin3 and RDP3/HDAC1 is displaced by a HATcontaining coactivator complex comprising CBP, p/CAF and SRC-1 (reviewed in (Xu et al., 1999).

Co-crystal structures have revealed that helix 12 of the ligand binding domain of the related estrogen receptor (also harbouring transrepressive properties) adopts a different conformation when bound to agonistic versus antagonistic estrogens (Brzozowski et al., 1998; Nichols, Rientjes and Stewart, 1998). Antagonist-bound progesterone receptor and estrogen receptor were further found to interact in vitro with the corepressors NcoR and SMRT (Jackson et al., 1997; Lavinsky et al., 1998; Wagner et al., 1998; Zhang et al., 1998). Multiple ligands for nuclear receptors are thus capable of influencing the biological activity of the receptor by selectively affecting the recruitment of specific cofactor complexes. The role of each cofactor in vivo can be assessed by knockout models of the individual cofactors, such as for SRC-1 (Xu et al., 1998) or by cell reconstitution experiments (Lemon et al., 2001). Undoubtedly, GR will also recruit its own specific cofactor configuration to enable transactivation. The question is whether there is also a role for corepressor molecules in transrepression mechanisms between NF-κB and GR? GR recruitment of HDAC2 was shown to inhibit interleukin-1β-induced histone acetylation at specific lysine residus (Ito, Barnes and Adcock, 2000). Furthermore, this GR association with HDAC2 in vivo could be disrupted by a GR antagonist, mifepristone (Ito et al., 2001). A deacetylase inhibitor trichostatine A (TSA) allegedly demonstrated involvement of histone deacetylase activities in GR-transrepression mechanisms. However, the relative transrepression levels in comparing TNF+DEX+TSA with TNF+TSA where identical to the ones observed without TSA. De facto, these data alone do not allow to conclude on whether deacetylases are involved in the mechanism of transrepression between GR and NF-κB (Vanden Berghe et al., 2002). In addition, promoter responsivity to TSA does not necessarily reflect sensitivity to GCs as both IL-8 and HIV promoter activities can be increased by TSA, whereas only IL-8 is responsive to GC repression (Vanden Berghe et al., 2002).

Future directions:

It was mentioned earlier that a GR-mediated Serine phosphorylation switch of RNA polymerase II could lie at the basis of NF-κB and GR cross-talk mechanisms (Nissen and Yamamoto, 2000). It would be of utmost interest to investigate whether this event is promoter-specific or a more general characteristic of GC-mediated repression of NF-κB. It would furthermore be interesting to know whether this mechanism could also account for the reciprocal mechanism. GR is also able to block phosphorylation of CBP, which could be another or additional way by which this transcription factor can modulate gene expression (Perissi et al., 1999). Involvement of this modification in cross-repression between NF-κB and GR remains to be explored. As not only histone tails but also nuclear receptors, NF-κB and cofactors can be acetylated or deacetylated, it will be interesting to learn how these various factor modifications can influence cross-talk mechanisms, in particular between GR and NF-κB. Similarly, other post-translational modifications may exert their influence. Hormone-dependent histone 3- or histone-4 specific methyltransferases (CARM1 and PRMT1 resp.) have now been characterized in transactivation (Ma et al., 2001; Wang et al., 2001). Again, not only histones but also CBP/p300 is targeted by CARM1, causing a disturbance of interaction with the transcription factor CREB (Ma et al., 2001). A link between these enzymatic activities and GR-NF-κB interplay has not yet been established but will probably be explored in the future. As pointed out above, chromatin components can be modified by diverse enzymatic activities. Another quite novel concept is the so-called chromatin remodelling, referring to the alteration in the chromatin fiber structure of a particular nucleosome, or a series of adjacent nucleosomes. To this extent, liganded nuclear receptors may associate with remodelling components SWI/SNF and utilize large ATP-dependent complexes (Kniyamu et al., 2000; Urnov and Wolffe, 2001) to bring about these structural changes before any other modifications involved in transcription initiation may occur. Whether the transrepression of p65 by GR and/or the reciprocal transrepression of GR by p65 could work through influencing the chromatinremodeling machinery needs further experimentation and confirmation in vivo. Finally, we would like to stress the fact that abovementioned models need not be exclusive. A direct protein-protein interaction would not rule out the involvement of cofactors or modulation by chromatin-or factor modifying enzymatic activities such as acetylation, methylation, ubiquitilation, etc. Understanding the subtle regulation and the precise contribution of each of these activities and parameters is of paramount importance to be able to design more specific anti-inflammatory strategies. The aim is to keep the already known great effectiveness of glucocorticoid hormones at place but to get rid of any detrimental side-effects associated with their long-term usage.

REFERENCES:

1. Abbinante-Nissen, J. M., Simpson, L. G. and Leikauf, G. D. (1995). Corticosteroids increase secretory leukocyte protease inhibitor transcript levels in airway epithelial cells. Am. J. phys. 268.

2. Adcock, I. M. and Caramori, G. (2001). Cross-talk between pro-inflammatory transcription factors and glucocorticoids. Immunology and Cell Biology 79, 376-384.

3. Adcock, I. M., Nasuhara, Y., Stevens, D. A. and Barnes, P. J. (1999). Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation: preferential targetting of NF-kappaB and lack of I-kappaB involvement. Br J Pharmacol 127, 1003-11.

4. Adcock, I. M., Newton, R. and Barnes, P. J. (1997). NF-kappa B involvement in IL-1 beta-induction of GM-CSF and COX-2: inhibition by glucocorticoids does not require I-kappa B. Biochem Soc Trans 25, 154s.

5. Amrani, Y., Lazaar, A. L. and Panettieri, R. A. J. (1999). Up-regulation of ICAM-1 by cytokines in human tracheal smooth muscle cells involves an NF-kappa B-dependent signaling pathway that is only partially sensitive to dexamethasone. J. Immunol. 163, 2128-2134.

6. Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A. and Karin, M. (1995). Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 270, 286-90.

7. Balsalobre, A., Brown, S. A., Marcacci, L., Tronche, F., Kellendonk, C., Reichardt, H. M., Schütz, G. and Schnibler U. (2000). Resetting of circadian time in peripheral tissues by glucocorticoid signalling. Science 289, 2344-2347.

8. Barnes, P. J. (1998). Anti-inflammatory actions of glucocorticoids: molecular mechanisms [editorial]. Clin Sci Colch 94, 557-72.

9. Barnes, P. J. and Adcock, I. M. (1997). NF-kappa B: a pivotal role in asthma and a new target for therapy. Trends Pharmacol Sci 18, 46-50.

10. Barnes, P. J., Greening, A. P. and Crompton, G. K. (1995). Glucocorticoid resistance in asthma. Am J Respir Crit Care Med 152, s125- 40.

11. Barnes, P. J. and Karin, M. (1997). Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N.Engl.J.Med 336, 1066-1071.

12. Beato, M. (1989). Gene regulation by steroid hormones. Cell 56, 335-344.

13. Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S. and Baltimore, D. (1995). Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376, 167-170.

14. Beyaert, R. (1999). NF-kappa B as an emerging target in atopy. Emerging Therapeutic Targets 3.

15. Beyaert, R., Suffys, P., Van Roy, F. and Fiers, W. (1990). Inhibition by glucocorticoids of tumor necrosis factor-mediated cytotoxicity. Evidence against lipocortin involvement. FEBS-Letters 262, 93-96.

16. Boumpas, D. T., Chrousos, G. P., Wilder, R. L., Cupps, T. R. and Balow, J. E. (1993). Glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates. Ann Intern Med 119, 1198-208.

17. Brostjan, C., Anrather, J., Csizmadia, V., Stroka, D., Soares, M., Bach, F. H. and Winkler, H. (1996). Glucocorticoid-mediated repression of NFkappaB activity in endothelial cells does not involve induction of IkappaBalpha synthesis. J Biol Chem 271, 19612-19616.

18. Brzozowski, A. M., Pike, A. C. W., Dauter, Z., Hubbard, R. E., bonn, T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J. A. and Carlquist, M. (1998). Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389, 753-758.

19. Burke, L. J. and Baniahmad, A. (2000). Corepressors. FASEB J. 14, 1876-1888.

20. Caelles, C., Gonzalez Sancho, J. M. and Muñoz, A. (1997). Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway. Genes Dev. 11, 3351-3364.

21. Caldenhoven, E., Liden, J., Wissink, S., Van de Stolpe, A., Raaijmakers, J., Koenderman, L., Okret, S., Gustafsson, J. A. and Van der Saag, P. T. (1995). Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids. Mol Endocrinol 9, 401-12.

22. Cato, A. C. and Wade, E. (1996). Molecular mechanisms of anti-inflammatory action of glucocorticoids. Bioessays 18, 371-8.

23. Chakravarti, D., LaMorte, V. J., Nelson, M. C., Nakajima, T., Schulman, I. G., Juguilon, H., Montminy, M. and Evans, R. M. (1996). Role of CBP/P300 in nuclear receptor signalling. Nature 383, 99-103.

24. Chaudhary, L. R. and Avioli, L. V. (1996). Regulation of interleukin-8 gene expression by interleukin-1beta, osteotropic hormones, and protein kinase inhibitors in normal human bone marrow stromal cells. J Biol Chem 271, 16591-6.

25. De Bosscher, K., Schmitz, M. L., Vanden Berghe, W., Plaisance, S., Fiers, W. and Haegeman, G. (1997). Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation. Proc Natl Acad Sci U S A 94, 13504-13509.

26. De Bosscher, K., Vanden Berghe, W. and Haegeman, G. (2000a). Mechanisms of anti-inflammatory action and of immunosuppression by glucocorticoids: negative interference of activated glucocorticoid receptor with transcription factors. J. Neuroimmunology 109, 16-22.

27. De Bosscher, K., Vanden Berghe, W. and Haegeman, G. (2001). Glucocorticoid repression of AP-1 is not mediated by competition for nuclear coactivators. Molecular Endocrinology 15, 219-227.

28. De Bosscher, K., Vanden Berghe, W., Vermeulen, L., Plaisance, S., Boone, E. and Haegeman., G. (2000b). Glucocorticoids repress NFkB- driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell. Proc. Natl. Acad. Sci. 97, 3919-3924.

29. De Kloet, E. R., Vreugdenhil, E., Oitzl, M. S. and Joels, M. (1998). Brain corticosteroid receptor balance in health and disease. Endocr Rev 19, 269-301.

30. De Vera, M. E., Taylor, B. S., Wang, Q., Shapiro, R. A., Billiar, T. R. and Geller, D. A. (1997). Dexamethasone suppresses iNOS gene expression by upregulating I-kappa B alpha and inhibiting NF-kappa B. Am J Physiol 273, g1290-6.

31. DeFranco, D. B. and Guerrero, J. (2000). Nuclear matrix targeting of steroid receptors: specific signal sequences and adaptor proteins. Crit. Rev. Eukaryot. Gene Expr. 10, 39-44.

32. Delany, A. M., Gabbitas, B. Y. and Canalis, E. (1995). Cortisol downregulates osteoblast alpha 1 (I) procollagen mRNA by transcriptional and posttranscriptional mechanisms. J Cell Biochem 57, 488-94.

33. Doucas, V., Shi, Y., Miyamoto, S., West, A., Verma, I. and Evans, R. M. (2000). Cytoplasmic catalytic subunit of protein kinase A mediates cross-repression by NF-kappa B and the glucocorticoid receptor. Proc. Natl. Acad. Sci 97, 11893-11898.

34. Doucas, V., Tini, M., Egan, D. A. and Evans, R. M. (1999). Modulation of CREB binding protein function by the promyelocytiv (PML) oncoprotein suggests a role for nuclear bodies in hormone signalling. Proc. Natl. Acad. Sci 96, 2627-2632.

35. Eggert, M., Mows, C. C., Tripier, D., Arnold, R., Michel, J., Nickel, J., Schmidt, S., Beato, M. and Renkawitz, R. (1995). A fraction enriched in a novel glucocorticoid receptor-interacting protein stimulates receptor-dependent transcription in vitro. J Biol Chem 270, 30755-9.

36. Francastel, C., Schubeler, D., Martin, D. I. and Groudine, M. (2000). Nuclear compartmentalization and gene activity. Nat. Rev. Mol. Cell Biol. 1, 137-143.

37. Gerritsen, M. E., Williams, A. J., Neish, A. S., Moore, S., Shi, Y. and Collins, T. (1997). CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci U.S.A. 94, 2927-2932.

38. Hager, G. L., Lim, C. S., Elbi, C. and Baumann, C. T. (2000). TRafficking of nuclear receptors in living cells. J. Steroid Biochem. Mol. Biol. 74, 249-254.

39. Heck, S., Bender, K., Kullmann, M., Gottlicher, M., Herrlich, P. and Cato, A. C. (1997). I kappaB alpha-independent downregulation of NF-kappaB activity by glucocorticoid receptor. EMBO J 16, 4698-707.

40. Heck, S., Kullmann, M., Gast, A., Ponta, H., Rahmsdorf, H. J., Herrlich, P. and Cato, A. C. (1994). A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1. EMBO J 13, 4087-4095.

41. Helmberg, A., Auphan, N., Caelles, C. and Karin, M. (1995). Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor. Embo J 14, 452-60.

42. Hinz, M., Krappmann, D., Eichten, A., Heder, A., Scheidereit, C. and Strauss, M. (1999). NF-kappa B function in growth control: regulation of cyclin D expression and G0/G1-to-S-phase transition. Mol Cell Biol 19, 2690-2698.

43. Hofmann, T. G., Hehner, S. P., Bacher, S., Droge, W. and Schmitz, M. L. (1998). Various glucocorticoids differ in their ability to induce gene expression, apoptosis and to repress NF-kappaB-dependent transcription. FEBS Lett 441, 441-6.

44. Horwitz, K. B., Jackson, T. A., Bain, D. I., Richer, J. K., Takimoto, G. K. and Tung, L. (1996). Nuclear receptor coactivators and corepressors. Mol Endocrinol 10, 1167-1177.

45. Ito, K., Barnes, P. J. and Adcock, I. M. (2000). Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits IL-1beta-induced histone H4 acetylation on Lysines 8 and 12. Mol. Cell. Biol. 20, 6891-6903.

46. Ito, K., Jazrawi, E., Cosio, B., Barnes, P. J. and Adcock, I. M. (2001). p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex. J. Biol. Chem. 276, 30208-30215.

47. Jackson, T. A., Richer, J. K., Bain, D. L., Takimoto, G. S., Tung, L. and Horwitz, K. B. (1997). The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11, 693-705.

48. Jehn, B. M. and Osborne, B. A. (1997). Gene regulation associated with apoptosis. Crit. Rev. Eukaryot.Gene Expr. 7, 179-193.

49. Kaltschmidt, B., Kaltschmidt, C., Hehner, S. P., Dröge, W. and Schmitz, M. L. (1999). Repression of NF-kappa B impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators. Oncogene 18.

50. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K. and Rosenfeld, M. G. (1996). A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85, 403-414.

51. Karin, M. (1998). New twists in gene regulation by glucocorticoid receptor: is DNA binding dispensable? Cell 93, 487-490.

52. Kassel, O., Sancono, A., Krätzschmar, J., Kreft, B., Stassen, M. and Cato, C. B. (2001). Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J 20, 7108-7116.

53. Kniyamu, H. K., Fryer, C. J., Horwitz, K. B. and Archer, T. K. (2000). The mouse mammary tumor virus promoter adopts distinct chromatin structures in hulan breast cancer cells with and without glucocorticoid receptor. J. Biol. Chem. 275, 20061- 20068.

54. Korzus, E., Torchia, J., Rose, D. W., Xu, L., Kurokawa, R., McInerney, E. M., Mullen, T. M., Glass, C. K. and Rosenfeld, M. G. (1998). Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 279, 703- 707.

55. Lasa, M., Brook, M., Saklatvala, J. and Clark, R. A. (2001). Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38. Mol. Cell. Biol. 21, 771-780.

56. Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen, T. M., Schiff, R., Del Rio, A. L., Ricote, M., Ngo, S., Gemsch, J., Hilsenbeck, S. G., Osborne, C. K., Glass, C. K., Rosenfeld, M. G. and Rose, D. W. (1998). Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci U S A 95, 2920-2925.

57. Lefstin, J. A. and Yamamoto, K. R. (1998). Allosteric effects of DNA on transcriptional regulators. Nature 392, 885-888.

58. Lemon, B., Inouye, C., King, D. S. and Tjian, R. (2001). selectivity of chromatin-remodelling cofactors for ligand-activated transcription. Nature 414, 924-928.

59. control. Genes Dev 14, 2551-2569.

60. Lezoualc'h, F., Sagara, Y., Holsboer, F. and Behl, C. (1998). High constitutive NF-kappaB activity mediates resistance to oxidative stress in neuronal cells. J Neurosci 18, 3224-32.

61. Liden, J., Delaunay, F., Rafter, I., Gustafsson, J. and Okret, S. (1997). A new function for the C-terminal zinc finger of the glucocorticoid receptor. Repression of RelA transactivation. J Biol Chem 272, 21467-72.

62. Liden, J., Rafter, I., Truss, M., Gustafsson, J. and Okret, S. (2000). Glucocorticoid effects on NF-kappa B binding in the transcription of the ICAM-1 gene. Biochem Biophys Res Commun 273, 1008-1014.

63. Ma, H., Baumann, C. T., Li, H., Strahl, B. D., Rice, R., Jelinek, M. A., Aswad, D. W., Allis, C. D. and Stallcup, M. R. (2001). hormonedependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter. Curr Biol. 11, 1981-1985.

64. McEwan, I. J., Wright, A. P. and Gustafsson, J. A. (1997). Mechanism of gene expression by the glucocorticoid receptor: role of protein-protein interactions. Bioessays 19, 153-160. McKay, L. I. and Cidlowski, J. A. (1998). Cross-talk between nuclear factor-kappa B and the steroid hormone receptors: mechanisms of mutual antagonism. Mol Endocrinol 12, 45-56.

65. Meyer, T., Carlstedt Duke, J. and Starr, D. B. (1997). A weak TATA box is a prerequisite for glucocorticoid-dependent repression of the osteocalcin gene. J Biol Chem 272, 30709-14.

66. Moras, D. and Gronemeyer, H. (1998). The nuclear receptor ligand-binding domain: structure and function. CURRENT.OPINION.IN CELL BIOLOGY. 10, 384-391.

67. Newton, R., Hart, L. A., Stevens, D. A., Bergmann, M., Donnelly, L. E., Adcock, I. M. and Barnes, P. J. (1998). Effect of dexamethasone on interleukin-1beta-(IL-1beta)-induced nuclear factor-kappaB (NF-kappaB) and kappaB-dependent transcription in epithelial cells. Eur J Biochem 254, 81-9.

68. Nichols, M., Rientjes, J. M. and Stewart, A. F. (1998). Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists. EMBO J 17, 765-73.

69. Nissen, R. M. and Yamamoto, K. R. (2000). The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev 14, 2314-2329.

70. Onate, S. A., Tsai, S. Y., Tsai, M. J. and O'Malley, B. W. (1995). Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270, 1354-1357.

71. Oro, A. E., Hollenberg, S. M. and Evans, R. M. (1988). Transcriptional inhibition by a glucocorticoid receptor-beta-galactosidase fusion protein. Cell 55, 1109-1114.

72. Perissi, V., Dasen, J. S., Kurokawa, R., Wang, Z. Y., Korzus, E., Rose, D. W., Glass, C. K. and Rosenfeld, M. G. (1999). Factorspecific modulation of CREB-binding protein acetyltransferase activity. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 96, 3652-3657.

73. Perkins, N. D. (1997). Achieving transcriptional specificity with NF-kB. Int J Biochem Cell Biol 29, 1433-1448.

74. Ramdas, J. and Harmon, J. M. (1998). Glucocorticoid-induced apoptosis and regulation of NF-kappaB activity in human leukemic T cells. Endocrinology 139, 3813-21.

75. Ray, K. P. and Searle, N. (1997). Glucocorticoid inhibition of cytokine-induced E-selectin promoter activation. Biochem Soc Trans 25, 189s.

76. Ray, P., Ghosh, S. K., Zhang, D. H. and Ray, A. (1997). Repression of interleukin-6 gene expression by 17 beta-estradiol: inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-kappa B by the estrogen receptor. FEBS Lett 409, 79- 85.

77. Reichardt, H. M., Kaestner, K. H., Tuckermann, J., Kretz, O., Wessely, O., Bock, R., Gass, P., Schmid, W., Herrlich, P., Angel, P. and Schutz, G. (1998). DNA binding of the glucocorticoid receptor is not essential for survival [see comments]. Cell 93, 531- 541.

78. Reichardt, H. M., Tuckermann, J. P., Göttlicher, M., Vujic, M., Weih, F., Angel, P., Herrlich, P. and Schütz, G. (2001). Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor. EMBO J 20, 7168-7173.

79. Resche-Rigon, M. and Gronemeyer, H. (1998). Therapeutic potential of selective modulators of nuclear receptor action. Curr. Op. Chem. Biol. 2, 501-507.

80. Scheinman, R., Cogswell, P. C., Lofquist, A. and Baldwin, A. S. (1995a). Role of transcriptional activation of i kappa b alpha in mediation of immunosuppression by glucocorticoids [see comments]. Science 270, 283-286.

81. Scheinman, R. I., Gualberto, A., Jewell, C. M., Cidlowski, J. A. and Baldwin, A. S., Jr. (1995b). Characterization of mechanisms involved in transrepression of NF- kappa B by activated glucocorticoid receptors. Mol Cell Biol 15, 943-953.

82. Schmitz, M. L., Stelzer, G., Altmann, H., Meisterernst, M. and Baeuerle, P. A. (1995). Interaction of the COOH-terminal transactivation domain of p65 NF-kappa B with TATA-binding protein, transcription factor IIB, and coactivators. J Biol Chem 270, 7219- 7226.

83. Sheppard, K.-A., Phelps, K. M., Williams, A. J., Thanos, D., Glass, C. K., Rosenfeld, M. G., Gerritsen, M. E. and Collins, T. (1998). Nuclear integration of glucocorticoid receptor and nuclear factor-kappaB signaling by CREB-binding protein and steroid receptor coactivator-1. J Biol Chem 273, 29291-4.

84. Sheppard, K.-A., Rose, D. W., Haque, Z. K., Kurokawa, R., McInerney, E., Westin, S., Thanos, D., Rosenfeld, M. G., Glass, C. K. and Collins, T. (1999). Transcriptional activation by NF-kappa B requires multiple coactivators. Mol Cell Biol 19, 6367-6378.

85. Stenoien, D. L., Mancini, M. G., Patel, K., Allegretto, E. A., Smith, C. L. and Mancini, M. A. (2000). Subnuclear trafficking of estrogen receptor alpha and steroid receptor coactivator-1. Mol. Endocrinol. 15, 518-534.

86. Stewart, S. and Crabtree, G. R. (2000). Transcription. Regulation of the regulators. Nature 408, 46-47.

87. Swantek, J. L., Cobb, M. H. and Geppert, T. D. (1997). Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alfa (TNF-a) translation: glucocorticoids inhibit TNF-afla translation by blocking JNK/SAPK. Mol Cell Biol 17, 6274-6282.

88. Tao, Y., Williams-Skipp, C. and Scheinman, R. I. (2001). Mapping of glucocorticoid receptor DNA binding domain surfaces contributing to transrepression of NF-kappaB and induction of apoptosis. J. Biol. Chem. 276.

89. Thiele, K., Bierhaus, A., Autschbach, F., Hofmann, M., Stremmel, W., Thiele, H., Ziegler, R. and Nawroth, P. P. (1999). Cell specific effects of glucocorticoid treatment on the NF-kappa Bp65/IkappaBalpha system in patients with Crohn's disease. Gut 45, 693-704.

90. Tobler, A., Meier, R., Seitz, M., Dewald, B., Baggiolini, M. and Fey, M. F. (1992). Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts. Blood 79, 45-51.

91. Unlap, M. T. and Jope, R. S. (1997). Dexamethasone attenuates NF-kappa B DNA binding activity without inducing I kappa B levels in rat brain in vivo. Brain Res Mol Brain Res 45, 83-9.

92. Urnov, F. D. and Wolffe, A. P. (2001). A necessary good: nuclear hormone receptors and their chromatin template. Mol. Endocrinol. 15, 1-16.

93. Vanden Berghe, W., De Bosscher, K., Plaisance, S., Boone, E. and Haegeman, G. (1999a). The Nuclear Factor-kappa B engages CBP/p300 and Histone Acetyltransferase Activity for Transcriptional Activation of the Interleukin-6 Gene Promoter. J Biol Chem 274, 32091-32098.

94. Vanden Berghe, W., De Bosscher, K., Vermeulen, L., De Wilde, G. and Haegeman, G. (2002). Induction and repression of NF-kappa B driven inflammatory genes. Springer-Verlag Series In Press.

95. Vanden Berghe, W., Francesconi, E., De Bosscher, K., Rèsche-Rigon, M. and Haegeman, G. (1998). Dissociated Glucocorticoids with Antiinflammatory Potential Repress Interleukin-6 Gene Expression by an NF-kappa-B-Dependent Mechanism. submitted .

96. Vanden Berghe, W., Francesconi, E., De Bosscher, K., Rèsche-Rigon, M. and Haegeman, G. (1999b). Dissociated Glucocorticoids with Antiinflammatory Potential Repress Interleukin-6 Gene Expression by an NF-kappa-B-Dependent Mechanism. Mol Pharmacol 56, 797-806.

97. Wagner, B. L., Norris, J. D., Knotts, T. A., Weigel, N. L. and McDonnell, D. P. (1998). The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol Cell Biol 18, 1369-78.

98. Wang, H., Huang, Z. Q., Xia, L., Feng, Q., Erdjument-Bromage, H., Strahl, B. D., Briggs, S. D., Allis, C. D., Wong, J., Tempst, P. and Zhang, Y. (2001). Methylation of Histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptors. Science 293, 853-857.

99. Wissink, S., van Heerde, E. C., Schmitz, M. L., Kalkhoven, E., van der Burg, B., Baeuerle, P. A. and van der Saag, P. T. (1997). Distinct domains of the RelA NF-kappaB subunit are required for negative cross-talk and direct interaction with the glucocorticoid receptor. J Biol Chem 272, 22278-84.

100. Wissink, S., van Heerde, E. C., vand der Burg, B. and van der Saag, P. T. (1998). A dual mechanism mediates repression of NF-kappaB activity by glucocorticoids. Mol Endocrinol 12, 355-63.

101. Wolffe, A. and Pruss, D. (1996). Targeting chromatin disruption: transcription regulators that acetylate histones. Cell 84, 817-819.

102. Wolffe, A. P. (1997). Transcriptional control. Sinful repression. Nature 387, 16-7.

104. Wolffe, A. P., wong, J. and Pruss, D. (1997). Activators and repressors: making use of chromatin to regulate transciption. Genes to Cells 2, 291-302.

105. Xu, J., Qiu, Y., DeMayo, F. J., Tsai, S., Tsai, M. and O'Malley, B. W. (1998). Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279, 1922-1925.

106. Xu, L., Glass, C. K. and Rosenfeld, M. G. (1999). Coactivator and corepressor complexes in nuclear receptor function. Curr Op Gen Dev 9, 140-147.

107. Young, J. D., Lawrence, A. J., MacLean, A. G., Leung, B. P., McInnes, I. B., Canas, B., Pappin, D. J. C. and Stevenson, R. D. (1999). Thymosin beta sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids. Nature Med 5, 1424-1427.

108. Zhang, X., Jeyakumar, M., Petukhov, S. and Bagchi, M. K. (1998). A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor. Mol Endocrinol 12, 513-24.

109. Zhong, H., Voll, R. E. and Ghosh, S. (1998). Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1, 661-671.

Received on 31.08.2011

Modified on 05.09.2011

Accepted on 08.09.2011

Research J. Pharmacology and Pharmacodynamics. 4(1):Jan. - Feb., 2012, 45-54